1. Field of the Invention
This invention relates to a method for immunoassay and reagents therefor, particularly to a method for immunoassay and a kit therefor which are suitable for quantitating a specific antigen or antibody present in a sample.
2. Related Art Statement
Radioimmunoassay (RIA) has heretofore been used in order to quantitate sample components with high sensitivity. However, RIA is disadvantageous in that handling of isotopes used therein such as their storage and disposal is troublesome, and therefore analytical methods which can be employed in place of RIA have been investigated.
For example, U.S. Pat. No. 4,483,921 discloses a method for immunoassay using lipid molecular membrane (as microcapsules, e.g., liposomes) having an antigen or antibody attached thereto.
An analytical method using microcapsules comprises encapsulating a labeling substance such as enzyme, fluorescent substance or luminous substance, and a precursor thereof in microcapsules, measuring the amount of the labeling substance released by destruction of the liposomes by antigen-antibody reaction, and thereby determining the concentration of a substance to be assayed.
As another example of this kind of analytical method, Japanese Patent Kokai (Laid-Open) No. 28661/84 discloses an immunoassay method comprising using sheep erythrocyte membrane as microcapsules, encapsulating an enzyme or a substrate into the microcapsules, and quantitating an antigen or an antibody by utilizing enzymatic reaction, and discloses reagents for immunoassay which are used for said immunoassay method.
In both of the methods described above, the same antigen or antibody as the substance to be assayed is attached to the surface of microcapsules. In all the conventional analytical methods using an antigen or antibody attached to microcapsules which is the same as a substance to be assayed, a so-called competitive reaction is utilized in which an antigen or antibody attached to microcapsules and an antigen or antibody in a sample react competitively with an antibody or antigen added as a reagent.
However, study by the present inventors has revealed that when a fat-soluble antigen is attached on microcapsules, the antigencity becomes lower in some cases.
Furthermore, when antigen is a protein, it is often attached on microcapsules by use of a bifunctional crosslinking agent such as glutaraldehyde, and it has been found that when attached on microcapsules by use of such a crosslinking agent, the protein is lowered in antigenic activity in some cases.
For these reasons, destruction of microcapsules (e.g., liposomes) by antigen-antibody reaction at the time of the competitive reaction becomes difficult to occur, resulting in a marked lowering of the detection sensitivity of assay and an increase of the required reaction time in some cases.
It has also been found that when the origin of an antigen attached to microcapsules is different from that of an antibody used for the reaction, the reaction activity and hence the detection sensitivity of assay are, in some cases, lower than when their origins are the same.
For example, when a substance to be assayed is one which is difficult to be synthesized or is per se expensive such as human insulin, an antigen from a different origin is often used in place of an antigen having the same origin. That is to say, in determining human insulin, bovine insulin can be attached to microcapsules and used for the reaction.
However, the following phenomenon has been confirmed by the present inventors. When the microcapsules having bovine insulin attached thereto and human insulin in a sample are competitively reacted with an anti-human insulin antibody which is highly reactive with human insulin in the sample, bovine insulin is less reactive with anti-human insulin antibody than human insulin, resulting in difficulty of destruction of the microcapsules.
On the other hand, as a method for immunoassay which is different from the competitive reaction method, Japanese Patent Kokai (Laid-Open) No. 269070/86 discloses a method which comprises covalently binding an antibody to liposomes encapsulating a labeling substance, reacting the resulting antibody-sensitized liposomes and free antibody with an antigen in a sample to obtain a sandwich type antigen-antibody complex, and thereby quantitating the antigen in the sample. However, this method is also disadvantageous in that the activity of the antibody bound to the liposomes is lowered, so that the determination sensitivity is lowered.
In addition, Japanese Patent Kokai (Laid-Open) No. 159652/85 discloses a method which comprises attaching a second antibody against a first antibody to microcapsules encapsulating a labeling substance, reacting the resulting second-antibody sensitized microcapsules with the first antibody and an antigen in a sample to obtain (antigen)-(first antibody)-(second-antibody)-sensitized microcapsules, adding thereto a complement, measuring the labeling substance released, and thereby quantitating the antigen in the sample. Such a method is disadvantageous in that since various reagents such as the first antibody and the second antibody should be prepared, the method is troublesome, and that since the method involves the two reaction steps, longer time is required for the determination of the antigen in the sample and further the determination sensitivity is lowered.